TWI718831B - Composition containing three extracts and their uses - Google Patents

Abstract

The present invention discloses that a composition comprising a solid phase extraction product of deep seawater, an extraction product of Roselle calyx, and an extraction product of Lactococcus lactis can be used to prevent and/or inhibit the growth of Streptococcus mutans, and to treat and/or Prevent the infection of Streptococcus mutans.

Description

Composition containing three extracts and their uses

The present invention relates to a composition for resistance to Streptococcus mutans , which comprises a solid phase extraction product of deep seawater, an extraction product of Hibiscus sabdariffa calyx (Hibiscus sabdariffa calyx), and lactic acid Extracted product of Lactococcus lactis. The present invention also relates to the composition can be used to prevent and/or inhibit the growth of Streptococcus mutans and treat and/or prevent infections of Streptococcus mutans.

Streptococcus mutans is a facultatively anaerobic gram-positive cocci, which is ubiquitous in human oral cavity and belongs to a common cariogenic bacteria. . When Streptococcus mutans begins to colonize the surface of teeth, the acidic substances produced in the process of metabolizing carbohydrates will corrode the enamel on the surface of the teeth, resulting in oral odor, dental caries, and dental caries. Occurrence of oral diseases such as gingivitis and dental calculus. In addition, Streptococcus mutans is also an opportunistic pathogen of infective endocarditis. It enters the bloodstream through wounds in the oral cavity, causing temporary bacteremia and adhesion. Vegetation is formed on the damaged heart valve to cause infective endocarditis.

Antibiotics currently used clinically to combat Streptococcus variabilis include ampicillin, cefotaxime, cefazolin, methicillin and clindamycin. However, the effects of these antibiotics against Streptococcus mutans are still not satisfactory. The main reasons include: the antibiotic resistance displayed by Streptococcus mutans and the serious side effects of antibiotics on patients. ) And adverse effects. Therefore, relevant researchers in the field try to find active components from natural sources that can be used to combat Streptococcus mutans.

There have been many studies on the application of extracts from plants or microorganisms in antibacterial. For example, in Alaga TO et al . (2014), J. Med. Plants Res. , 8:339-344, Alaga TO et al. soaked the calyces of Hibiscus sabdariffa in deionized water and After 12 hours in ethanol, it was filtered and the filtrate was collected, and the water-extracted product and the ethanol-extracted product of Roselle calyx were obtained. Next, Alaga TO et al. used these products for phytochemical analysis and identified them as containing saponins, alkaloids, tannins, total phenols, Flavonoids and glycosides. These products were confirmed to have antibacterial activity against pathogenic bacteria (including Streptococcus mutans) through disc and agar well diffusion methods.

In Fatemeh Babadi et al . (2018), J. Res. Med. Dent. Sci. , 6:198-202, Fatemeh Babadi et al. inoculated Lactobacillus acidophilus into MRS broth (MRS broth). ), the resulting culture was mixed with ethyl acetate (ehtyl acetate) and settled, then the organic layer was collected and the ethyl acetate was removed to obtain the Lactobacillus acidophilus metabolite ( Lactobacillus acidophilus metabolites). Afterwards, Fatemeh Babadi et al. took the Lactobacillus acidophilus metabolites for an anti-oral streptococci test, and found that the Lactobacillus acidophilus metabolites had antibacterial activity against oral streptococci.

In addition, TW 201924697 A reveals a deep seawater organic extract, which contains solid organic matter. The solid organic matter is prepared by using C18 filter membrane to extract deep seawater. The deep seawater organic extract has been proved to have the effect of inhibiting the growth of Helicobacter pylori through experiments.

After research, the applicant unexpectedly found that a composition containing solid-phase extraction products of deep seawater, extracts of Roselle calyx, and extracts of Lactococcus lactis can effectively prevent and/or inhibit the growth of Streptococcus mutans. Therefore, it is expected to be used for the treatment and/or prevention of Streptococcus mutans infection.

Summary of the invention

Therefore, in the first aspect, the present invention provides a composition for resistance to Streptococcus mutans, which comprises a solid phase extraction product of deep seawater, an extraction product of Roselle calyx, and an extraction product of Lactococcus lactis.

In a second aspect, the present invention provides a use of the composition as described above for preparing a medicine for preventing and/or inhibiting the growth of Streptococcus mutans.

In a third aspect, the present invention provides a method for preventing and/or inhibiting the growth of Streptococcus mutans in an individual, which comprises administering a composition as described above to the individual.

In a fourth aspect, the present invention provides a use of the composition as described above for preparing a medicine for treating and/or preventing infection of Streptococcus mutans.

In a fifth aspect, the present invention provides a method for treating and/or preventing a Streptococcus mutans infection in an individual, which comprises administering to the individual a composition as described above.

Detailed description of the invention

It should be understood that if any previous case publication is quoted here, the previous case publication does not constitute a recognition: in Taiwan or any other country, the previous case publication forms a common general in the art. Part of knowledge.

For the purpose of this specification, it will be clearly understood that the word "comprising" means "including but not limited to", and the word "comprises" has a corresponding meaning.

Unless otherwise defined, all technical and scientific terms used in this article have meanings commonly understood by those familiar with the art of the present invention. A person familiar with the art will recognize that many methods and materials similar or equivalent to those described herein can be used to implement the present invention. Of course, the present invention is by no means restricted by the described methods and materials.

In the development of drugs that can be used against Streptococcus mutans ( Streptococcus mutans ), the applicant has discovered through experiments: a solid phase extraction product containing deep seawater, Hibiscus sabdariffa calyx The mixture of extraction products and Lactococcus lactis (Lactococcus lactis) extraction products can effectively inhibit the growth of Streptococcus mutans, and this inhibitory effect is significantly better than any one of the above three extraction products or a combination of any two. Therefore, it is expected to have a high potential for treating and/or preventing infections of Streptococcus mutans.

Therefore, the present invention provides a composition for resistance to Streptococcus mutans, which comprises a solid phase extraction product of deep seawater, an extraction product of Roselle calyx, and an extraction product of Lactococcus lactis.

Preferably, the extraction product of the Lactococcus lactis, the extraction product of the roselle calyx and the solid phase extraction product of the deep seawater are in a range of 1:1:4 (w/w/w) to 3:4 : Ratio within 7 (w/w/w). In a preferred embodiment of the present invention, the extraction product of the Lactococcus lactis, the extraction product of the roselle calyx and the solid phase extraction product of the deep seawater have a ratio of 1:1:3 (w/w/w). )proportion.

According to the present invention, the solid phase extraction product of the deep seawater has a content ranging from 5 to 100 mg/mL. Preferably, the solid phase extraction product of the deep seawater has a content ranging from 5 to 20 mg/mL. In a preferred embodiment of the present invention, the composition contains 16 mg/mL of the solid phase extraction product of deep seawater.

According to the present invention, the extract from the calyx of Roselle Seed has a content ranging from 1 to 80 mg/mL. Preferably, the extract from the calyx of Roselle Seed has a content ranging from 2 to 30 mg/mL. In a preferred embodiment of the present invention, the composition contains 3 mg/mL roselle calyx extract.

According to the present invention, the extracted product of Lactococcus lactis has a content ranging from 1 to 50 mg/mL. Preferably, the extracted product of Lactococcus lactis has a content ranging from 1 to 20 mg/mL. In a preferred embodiment of the present invention, the composition contains 1 mg/mL Lactococcus lactis extract.

According to the present invention, the solid phase extraction product of the deep seawater is prepared by performing a C18 solid phase extraction on the deep seawater to remove ionic compounds.

As used herein, the terms "deep sea water" and "marine deep water" are used interchangeably, and mean a collection from a specific depth below sea level Sea water, it has the characteristics of low temperature, cleanness, and nutrient salt-rich. Preferably, the deep seawater is taken from seawater falling within a depth of 300 to 600 meters in a range below sea level. In a preferred embodiment of the present invention, the deep seawater is taken from seawater at a depth of 200 meters below sea level.

As used herein, the term "solid phase extraction" means a chemical separation technique that dissolves or suspends a sample in a liquid [known as mobile phase (mobile phase) )] and flow through a solid [called a stationary phase or sorbent], and then use the difference in the affinity of each component in the sample for the solid to be separated into Desired and undesirable components. In some embodiments, the desired components are retained on the adsorbent. In some specific cases, undesired components are retained on the adsorbent. In a preferred embodiment of the present invention, the desired components are retained on the adsorbent.

As used herein, the term "C18 solid phase extraction" means solid phase extraction using C18 material as an adsorbent. Preferably, C18 solid phase extraction uses columns, tubes, syringes, cartridges and disks containing C18-bonded silica particles (C18-bonded silica particles). (discs) and was carried out.

According to the present invention, the C18 solid-phase extraction can be performed using a technique well-known and commonly used by those skilled in the art. In this regard, you can refer to, for example, TW 201924697 A, US 2016/0282371 A1, and US 2013/0261315 A1.

It can be understood that the operating conditions related to solid phase extraction will be further changed with factors such as the source and total amount of the sample used in order to achieve the best extraction effect. The choice of these operating conditions is routinely determined by those who are familiar with the art.

According to the present invention, the solid phase extraction product of the deep seawater contains 9,000-13,000 ppm sodium, 150-600 ppm potassium, 210-700 ppm calcium, and 640-1,800 ppm magnesium. In a preferred embodiment of the present invention, the solid phase extraction product of the deep seawater contains 12,100 ppm of sodium, 443 ppm of potassium, 427 ppm of calcium, and 1,430 ppm of magnesium.

According to the present invention, the solid phase extraction product of the deep seawater contains 5-70% organic substances with a molecular weight in the range of 50 to 700 Da. In a preferred embodiment of the present invention, the solid phase extraction product of the deep seawater contains 50% organic substances with a molecular weight in the range of 200 to 700 Da.

According to the present invention, the extraction product of the calyx of Roselle and the extraction product of Lactococcus lactis can be carried out using extraction techniques that are well-known and commonly used by those skilled in the art. In this regard, one can refer to, for example, Fatemeh Babadi et al . (2018) (same as above).

It can be understood that the extraction operating conditions will be further changed with factors such as the type and amount of extraction solvent used in order to achieve the best extraction effect. The choice of these operating conditions is routinely determined by those who are familiar with the art.

Preferably, the extraction product of the calyx of Roselle is prepared by extracting the calyx of Roselle using a solvent selected from the group consisting of: ethyl acetate, ethyl acetate, Methanol, ethanol, water, and combinations thereof. In a preferred embodiment of the present invention, the extraction product of the roselle calyx is a product extracted by ethyl acetate.

Preferably, the extraction product of Lactococcus lactis is prepared by using a solvent selected from the group consisting of the following to extract the culture of Lactococcus lactis: ethyl acetate, n-hexane (n-hexane), and their combination. In a preferred embodiment of the present invention, the extraction product of Lactococcus lactis is a product extracted with ethyl acetate.

More preferably, the extraction product of the calyx of Roselle Seed is prepared by the following conditions: Let the range fall from 1:10 (w/v, g/mL) to 1:20 (w/v, g/mL) The amount of roselle calyx and ethyl acetate in the ratio within) are mixed under a pressure ranging from -50 to 0 kpa and a temperature ranging from 15 to 40°C for 5 to 12 hours.

More preferably, the extraction product of Lactococcus lactis is prepared under the following conditions: a range of lactic acid milk in an amount ratio ranging from 1:5 (v/v) to 1:20 (v/v) The culture of cocci and ethyl acetate are mixed at a temperature ranging from 15 to 40°C for 5 to 12 hours.

According to the present invention, the ethyl acetate-extracted product of Roselle's calyx contains 1-30% anthocyanin, 10-70% polyphenol, and 5-10% organic acid. acid) and 5-10% amino acid. Preferably, the anthocyanins include, but are not limited to: cyanidin, delphinidin, malvidin, pelargonidin, peonidin, and Morning glory (petunidin).

According to the present invention, the ethyl acetate-extracted product of Lactococcus lactis contains antimicrobial peptides (AMPs). Preferably, the antibacterial peptide includes, but is not limited to: nisin, lacticin, carnobacteriocin, enterocin and pediocin . In a preferred embodiment of the present invention, the ethyl acetate-extracted product of Lactococcus lactis contains nisin.

According to the present invention, the mixture containing three kinds of extracts has been confirmed to be able to effectively inhibit the growth of Streptococcus var. var. strains through the determination of the minimum inhibitory concentration (minimum inhibitory concentration). Accordingly, the mixture containing the three extracts according to the present invention is expected to have antibacterial activity, and thus can be applied to the preparation of a medicine for preventing and/or inhibiting the growth of Streptococcus mutans. In addition, the present invention also provides a method for preventing and/or inhibiting the growth of Streptococcus mutans in an individual, which comprises administering the above-mentioned drug to the individual.

Based on the above-mentioned antibacterial activity, the mixture containing 3 kinds of extracts according to the present invention is expected to be applied to the preparation of a medicine for treating and/or preventing infection of Streptococcus mutans. The present invention also provides a method for treating and/or preventing Streptococcus mutans infection in an individual, which comprises administering the above-mentioned medicine to the individual.

As used herein, the term "treating" or "treatment" of an infection of Streptococcus var. var. means that the severity of the infection or the symptoms of the infection are reduced, Either the infection is partially or completely eliminated (eliminated).

As used herein, the term “preventing” or “prevention” of an infection of Streptococcus var. var. means that an individual eliminates or reduces the infection when the infection has not been diagnosed. The incidence of infection, and slow, delay, control or decrease the likelihood or probability of the infection.

According to the present invention, the medicine can be manufactured into a dosage form suitable for parenteral administration, oral administration, or topical administration by using techniques well known to those skilled in the art. dosage form).

According to the present invention, the drug may further include a pharmaceutically acceptable carrier widely used in drug manufacturing technology. For example, the pharmaceutically acceptable carrier may include one or more reagents selected from the group consisting of solvents, buffers, emulsifiers, suspending agents, decomposers ), disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent , Gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome and the like. The selection and quantity of these reagents fall within the scope of professionalism and routine techniques of those who are familiar with this technology.

According to the present invention, the drug can be administered by a parenteral route selected from the group consisting of intraepidermal injection, intralesional injection, and sublingual injection. administration). Preferably, the medicine is manufactured in a dosage form suitable for sublingual administration.

According to the present invention, the medicine can be manufactured into a dosage form suitable for oral administration using techniques well-known to those skilled in the art. This includes, but is not limited to: sterile powder, tablet, and troche. ), lozenge, pellet, capsule, dispersible powder or granule, solution, suspension, emulsion, syrup , Elixir, slurry and the like. Preferably, the medicine is manufactured as an oral hygiene product, which includes, but is not limited to: chewing gums, dentifrices, mouthwash, mouth sprays ), buccal tablet, sustained film coated table and oral ointment.

According to the present invention, the medicine can also be manufactured into an external preparation suitable for topical application to the skin by using techniques well known to those skilled in the art. This includes, but is not limited to: emulsions. , Gel, ointment, cream, patch, liniment, powder, aerosol, spray, lotion , Serum, paste, foam, drop, suspension, salve and bandage.

The dosage and frequency of administration of the mixture containing the three extracts according to the present invention will vary depending on the following factors: the severity of the disease to be treated, the route of administration, and the age, physical condition and response of the individual to be treated. Generally speaking, the pharmaceuticals according to the present invention can be in the form of a single dose or divided into several doses, and can be administered parenterally, orally or locally.

Detailed description of the preferred embodiment

The present invention will be further described with respect to the following embodiments, but it should be understood that these embodiments are for illustrative purposes only, and should not be construed as limitations on the implementation of the present invention. Examples Example 1. Preparation of a mixture of three extracts of the present invention. Experimental materials: 1. Preparation of solid phase extraction product of deep seawater:

First, the deep seawater taken from Qixingtan in Hualien County and located 200 meters below sea level is sequentially treated with four 0.22 μm filter membranes (brand: KHAN, model: PUP-001-DOE-20-V) Filter to remove sand particles and microorganisms in the deep seawater. The obtained filtrate was determined to contain 12,100 ppm sodium, 443 ppm potassium, 427 ppm calcium, and 1,430 ppm magnesium through trace element analysis using an inductively coupled plasma (ICP) analyzer.

Then, 1.5 metric tons of filtrate was collected and a C18 solid phase extraction membrane (3M™ Empore™ C18 Extraction Disks) (product number 2215 or 2315, brand name 3M) was used for solid phase extraction. After that, 200 mL of 100% ethanol was added to elute the substances adsorbed on the C18 solid phase extraction membrane, and then the ethanol was removed by concentration under reduced pressure, and then dried to obtain a powder The solid-phase extraction product of deep seawater (hereinafter referred to as DSW extract).

Afterwards, the DSW extract was dissolved in deionized water to prepare a sample of the solution to be tested with a concentration of 10 mg/mL, and used to perform liquid chromatography-tandem mass spectrometry (liquid chromatography-tandem mass spectrometry). tandem mass spectrometry, LC-MS/MS). The LC-MS/MS analytical instruments used are as follows: ultra performance liquid chromatography (ultra performance liquid chromatography, UPLC) (model LC-30AD, brand Shimadzu) and quadrupole mass spectrometer (model It is API 4500, and the brand is AB SCIEX). The various operating parameters and conditions of LC-MS/MS are shown in Table 1 below. Table 1. Operating parameters and conditions of LC-MS/MS Operating parameters condition Separation column C18 pipe string (the article number is 00A-4461-AN, the brand is Kinetex) String specifications 30 mm × 2.1 mm Detection range 50-700 m/z Mobile phase Methanol/water, 100:0 (v/v) Gradient elution of mobile phase From 0 to 3 minutes, methanol changes from 100% to 70%; from 4 to 6 minutes, methanol changes from 70% to 20%; from 6 to 8 minutes, methanol changes from 20% to 0% ; At 8 to 10 minutes, methanol changes from 0% to 100%. Flow rate (mL/min) 0.4

Figure 1 shows the mass spectrum obtained by using the DSW extract for LC-MS/MS. It can be seen from Fig. 1 that the solid-phase extraction product of the deep seawater contains at least 12 different organic substances, and 50% of the organic substances have a molecular weight ranging from 200 to 700 Da. 2. Preparation of ethyl acetate-extracted product of Hibiscus sabdariffa calyx (ethyl acetate-extracted product of Hibiscus sabdariffa calyx):

First, the calyx of the fresh Hibiscus sabdariffa (Hibiscus sabdariffa) purchased from Jinfeng Township, Taitung County was washed with deionized water and crushed with a homogeneous crusher (model B96W-7010S, brand WARING) , And then freeze-drying lasted 72 hours. Then, the obtained roselle calyx with a water content of less than 3% is placed in a powder homogenizer (model D9KC-CK100, brand CHEMIST) for grinding, to obtain roselle calyx powder.

After that, 100 g of Roselle calyx powder was added to 1,000 mL of ethyl acetate and an ultrasonic processor (model O-LEO- 803, the brand is LEO ULTRASONIC) to vibrate for 8 hours, and then filter with a No. 1 filter paper with a pore size of 11 µm (the article number is AW1001-00090, the brand is Whatman), and the filtrate is collected and performed at 10,000 rpm Centrifugation lasted 20 minutes. Then, the supernatant was collected and filtered with the No. 1 filter paper, and then the collected filtrate was freeze-dried at -80°C to obtain a powdered roselle calyx which was extracted with ethyl acetate.的产品 (hereinafter referred to as HS extract).

After that, the HS extract was dissolved in ethyl acetate to prepare a sample of the solution to be tested with a concentration of 10 mg/mL, and proceeded roughly in accordance with the operating parameters and conditions described in item 1 above. The difference of LC-MS/MS is that the mobile phase uses acetonitrile instead of methanol, the detection range is adjusted to 50-400 m/z, and the flow rate is adjusted to 0.3 mL/min.

According to the results of LC-MS/MS, the ethyl acetate-extracted product of the roselle calyx contains 30% anthocyanin, 60% polyphenol, and 5% organic acid. ) And 5% amino acid. 3. Preparation of ethyl acetate-extracted product of Lactococcus lactis :

First, the Lactococcus lactis BCRC 10791 purchased from the Biosource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) in Taiwan was inoculated into the glucose broth Glucose broth medium (purchased from Difco) [contains casein enzymic hydrolysate, glucose and sodium chloride], and set a temperature of 30 ℃ The culture was carried out in the incubator for 48 hours, followed by freezing treatment at -80°C and thawing treatment at room temperature, and then centrifugation at 12,000 rpm for 20 minutes. Then, collect the supernatant and filter it with a filter paper with a pore size of 0.22 µm (the article number is 10116620, the brand is Merck), and then collect 100 mL of the filtrate and add it to 1,000 mL of ethyl acetate. After shaking at a temperature of 30°C for 8 hours, it was placed at -80°C for freeze-drying. After that, the obtained freeze-dried Lactococcus lactis (with a water content of less than 3%) is placed in a high-efficiency nanogrinding machine (model 3070506, brand PMC) for grinding treatment, thereby obtaining a A powdered product of Lactococcus lactis extracted with ethyl acetate (hereinafter referred to as LL extract).

After that, the LL extract was dissolved in ethyl acetate to prepare a test solution sample with a concentration of 10 mg/mL, and proceeded roughly in accordance with the operating parameters and conditions described in item 1 above. The difference of LC-MS/MS is that the mobile phase uses acetonitrile instead of methanol, the detection range is adjusted to 1111-1119 m/z, and the flow rate is adjusted to 0.3 mL/min. In addition, for comparison, nisin (product number N5764, brand Sigma-Aldrich) was used as a control standard and the same LC-MS/MS analysis was performed.

According to the results of LC-MS/MS, the ethyl acetate-extracted product of Lactococcus lactis contains nisin [including nisin complex], which belongs to an antimicrobial peptide (antimicrobial peptides). , AMPs). experimental method:

The powdered DSW extract, HS extract, and LL extract obtained from items 1 to 3 of the above "experimental materials" are given in a ratio of 3:1:1 (w/w/w) Mix uniformly, thereby obtaining a mixture of the present invention containing 3 kinds of extracts (hereinafter referred to as DSW-HS-LL mixture) for subsequent experiments. Example 2. Evaluation of the effectiveness of the mixture of the present invention containing 3 kinds of extracts against Streptococcus mutans:

In this example, the applicant used the DSW-HS-LL mixture obtained in Example 1 above to inhibit the growth of 50% of Streptococcus var. strain (minimum inhibitory concentration reached by 50%, MIC ₅₀ ) To evaluate its antibacterial activity against Streptococcus mutans (antibacterial activity). In addition, for comparison, the three extracts (that is, DSW extract, HS extract, and LL extract) obtained in the "experimental materials" of Example 1 above were used for the same experiment. Experimental materials: 1. Preparation of test bacterial solution of Streptococcus mutans:

First, the Streptococcus mutans BCRC 10793 purchased from the Biological Resources Conservation and Research Center of the Food Industry Development Institute in Taiwan was inoculated into the brain heart infusion (BHI) medium (purchased from Difco) [containing Beef heart, calf brains, disodium hydrogen phosphate, peptone, and glucose] were cultured in an incubator set at 30°C for 24 hour. After that, the formed culture was centrifuged at 5,000 rpm for 10 minutes. After pouring the supernatant solution, wash the pellets with phosphate-buffered saline (PBS), and then use BHI medium to fully suspend the bacteria and _{adjust the OD 600} value to 1, by In this way, a test bacterial solution of Streptococcus mutans (bacteria concentration is about 1×10 ⁷ CFU/mL) is obtained. experimental method:

First, the DSW-HS-LL mixture, DSW extract, HS extract, and LL extract obtained in Example 1 above were dissolved in deionized water and each prepared into a reserve with a concentration of 50 mg/mL. Stock solution. Then, the stock solutions were diluted with deionized water to obtain test solutions with different extract concentrations (0.5, 1, 2, 5, 10, 20, and 50 mg/mL).

After that, each test solution (2 mL) was added to each well of the 96-well plate containing 10 mL of the test bacterial solution prepared according to item 1 of the "Experimental Materials" above, Then, it was allowed to stand for 24 hours at 30°C. In addition, the test bacteria solution that was not treated with the test solution was used as a control.

After that, use an ELISA reader (model UVM340, brand Biochrom Asys) at a wavelength of 600 nm to read the absorbance value (OD ₆₀₀ ) of the bacterial culture formed in each well, and the MIC _{50 is} the The extract can reduce the OD ₆₀₀ value of the bacterial culture by more than 50% (compared to the lowest concentration of the control bacterial liquid phase). result:

_{The measured MIC 50} of the DSW-HS-LL mixture, DSW extract, HS extract, and LL extract for Streptococcus var. var. species were 5, 10, 20, and 20 mg/mL, respectively. The results of this experiment show that the combination of these 3 types is compared to any of the solid-phase extraction product of deep seawater, the ethyl acetate extraction product of Roselle's calyx, and the ethyl acetate extraction product of Lactococcus lactis The extract mixture obtained from the extract has obviously excellent antibacterial activity against Streptococcus mutans, and is therefore expected to have the highest potential for development against Streptococcus mutans. Example 3. Comparison of the effectiveness of different combinations of extracts on resistance to Streptococcus mutans:

_{In this example, the applicant selected the measured MIC 50} (ie 5 mg/mL) of the mixture of the three extracts of the present invention to compare the effectiveness of the combination of different extracts against Streptococcus mutans. Experimental materials: 1. Prepare an extraction mixture containing 2 kinds of extracts:

Mix any two of the DSW extract, HS extract, and LL extract obtained from the "experimental materials" of Example 1 above at a ratio of 1:1 (w/w) to obtain Three different mixtures (hereinafter referred to as DSW-HS mixture, DSW-LL mixture and HS-LL mixture). experimental method:

First, dissolve the DSW extract, HS extract, LL extract, DSW-HS mixture, DSW-LL mixture, HS-LL mixture, and DSW-HS-LL mixture in deionized water respectively to prepare a concentration of 5 mg/mL test solution.

After that, each test solution (2 mL) was added to each well of the 96-well culture plate containing 10 mL of the test bacterial solution prepared according to item 1 of the "experimental material" of Example 2 above, and then The static culture at 30°C lasted 24 hours. In addition, the test bacteria solution that was not treated with the test solution was used as a control. After that, an ELISA reader was used to read the absorbance (OD ₆₀₀ ) of each well at a wavelength of 600 nm.

The bacterial growth inhibition rate (%) is _{calculated by substituting the measured absorbance (OD 600} ) into the following formula (1): formula (1) : A=(C- B) / C × 100 where: a = bacterial growth inhibition rate (%) B = after processing to each test solution was measured by the OD absorbance of ₆₀₀ C = control OD ₆₀₀ absorbance bacteria measured if the measured The higher the bacterial growth inhibition rate, the higher the resistance of the extract to Streptococcus mutans. result:

Figure 2 shows the measured bacterial growth inhibition rate (%) of different combinations of extracts against Streptococcus variabilis. It can be seen from Figure 2 that the bacterial growth inhibition rate measured by DSW-HS-LL mixture is significantly higher than DSW extract, HS extract, LL extract, DSW-HS mixture, DSW-LL mixture and HS-LL mixture Possessed. The results of this experiment show the growth inhibitory effect of the combination of the solid-phase extraction product of deep seawater, the ethyl acetate extraction product of Roselle's calyx, and the ethyl acetate extraction product of Lactococcus lactis It is significantly better than either one or a combination of the two. Therefore, the mixture containing 3 kinds of extracts according to the present invention is expected to be useful for treating and/or preventing infections of Streptococcus mutans.

All patents and documents cited in this specification are incorporated into this case as reference materials in their entirety. If there is a conflict, the detailed description of the case (including the definition) will prevail.

Although the present invention has been described with reference to the above specific specific examples, it is obvious that many modifications and changes can be made without departing from the scope and spirit of the present invention. Therefore, it is intended that the present invention is only limited by the scope of the patent application attached hereto.

The above and other objectives, features and advantages of the present invention will become apparent with reference to the following detailed description and preferred embodiments and the accompanying drawings, in which: Figure 1 shows the mass spectrum obtained by using the DSW extract for LC-MS/MS; and Figure 2 shows the measured bacterial growth inhibition rate (%) of different combinations of extracts against Streptococcus variabilis.

Claims (6)

A composition for resistance to Streptococcus mutans, which contains a solid phase extraction product of deep seawater, an extraction product of Roselle calyx and an extraction product of Lactococcus lactis, wherein the solid phase extraction product of the deep seawater is obtained by The seawater was subjected to a C18 solid-phase extraction to remove ionic compounds. The extraction product of the roselle calyx was prepared by using ethyl acetate to extract the roselle calyx, and the Lactococcus lactis The extraction product is prepared by extracting the culture of Lactococcus lactis using ethyl acetate. The composition of claim 1, wherein the solid phase extraction product of the deep seawater has a content ranging from 5 to 100 mg/mL. The composition according to claim 1, wherein the extract of the calyx of Roselle has a content ranging from 1 to 80 mg/mL. The composition according to claim 1, wherein the extract product of Lactococcus lactis has a content ranging from 1 to 50 mg/mL. A use of the composition as claimed in any one of claims 1 to 4 for preparing a medicine for preventing and/or inhibiting the growth of Streptococcus mutans. A use of the composition as claimed in any one of claims 1 to 4 for preparing a medicine for treating and/or preventing infection of Streptococcus mutans.

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